J. Nat. Prod. 1999, 62, 873-876
873
Six Triterpenoid Saponins from Maesa laxiflora Zhong Jiang,† Jean-Franc¸ ois Gallard,† Marie-The´re`se Adeline,† Vincent Dumontet,† Mai Van Tri,‡ Thierry Se´venet,† and Mary Paı¨s*,† Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette, Cedex, France, and Institut de Chimie, NCST, Nghia Do, Cau Giay, Hanoi, Vietnam Received January 22, 1999
Six new triterpenoid saponins, maelaxins A-F (1-6), were isolated from a n-BuOH extract of the leaves of Maesa laxiflora. They possess 22-O-angeloyl-camelliagenin A, 16-O-acetyl, 22-O-angeloyl-camelliagenin A, or 22-O-(2Z)-hexenoyl-camelliagenin A as the aglycon. The pentasaccharide moiety linked to C-3 of the aglycon consists of D-glucuronic acid, L-rhamnose, D-glucose, and/or D-galactose. Their structures were elucidated by extensive NMR experiments including 1H-1H (COSY, 2D HOHAHA, NOESY) and 1H-13C (HMQC and HMBC) spectroscopy and chemical evidence. The saponins so far isolated from the genus Maesa show virucidal activity, fungistatic and antimutagenic properties,1 and piscicidal activity.2 Maesa laxiflora Pitard (Myrsinaceae), collected in the framework of a collaboration program between CNRS (France) and NCST (Vietnam), is a small tree that grows in Vietnam and southern regions of the People’s Republic of China, where it is used as a traditional medicine. Preliminary biological tests indicated that the EtOH extract of the leaves of Maesa laxiflora exhibited moderate cytotoxic activity (IC50 10 µg/mL) against A-549 cells and inhibition of the assembly of tubulin into microtubules (41%, 10 mg/mL). Our investigation on the n-BuOH extract of the leaves of this plant led to the isolation of six new triterpenoid saponins, named maelaxins A-F (1-6), respectively. This paper describes the isolation and structure elucidation of these six new saponins. All of the isolated saponins possess a pentasaccharide moiety linked to C-3 of the aglycon. Results and Discussion The EtOH extract of dried leaves of M. laxiflora was successively defatted with heptane and EtOAc, then partitioned between n-BuOH and water. The n-BuOH-soluble fraction was repeatedly subjected to Si gel chromatography to furnish the crude saponins. Reversed-phase HPLC gave six pure saponins, maelaxins A-F (1-6). Maelaxin A (1) was obtained as a white amorphous powder. The molecular formula was established as C65H104O29 on the basis of 13C NMR, 13C DEPT NMR, and MS. Positive-ion FABMS and ESMS gave a [M + 2Na - H]+ ion at m/z 1393 and a [M + Na]+ ion at m/z 1371. The negative-ion FABMS showed a [M - H]- anion at m/z 1347. Prominent fragment peaks occurred at m/z 1201 [M - H - 146]- and 1185 [M - H - 162]- due to the independent losses of a deoxyhexose and a hexose unit, indicating a branched oligosaccharide with terminal deoxyhexose and hexose. Other peaks were observed at m/z 1055 [M - H 146 - 146]-, 893 [M - H - 146 - 146 - 162]-, 731 [M H - 146 - 146 - 162 - 162]-, and 555 [M - H - 146 146 - 162 - 162 - 176]-, corresponding to the subsequent loss of a deoxyhexose and a hexose unit and finally a hexuronic acid unit. The peaks at m/z 1247 [M - H - 100]* To whom correspondence shoud be addressed. Tel.: +33 1 69 82 30 90. Fax: +33 1 69 07 72 47. E-mail:
[email protected]. † Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-surYvette, Cedex, France. ‡ Institut de Chimie, NCST, Nghia Do, Cau Giay, Hanoi, Vietnam.
10.1021/np990028s CCC: $18.00
and 793 [M - H - 146 - 146 - 162 - 100]- were attributed to the loss of an angeloyl or tigloyl group.3,4 The 13C NMR spectrum of 1 showed 65 signals, of which 35 were assigned to a substituted triterpenoid moiety and 30 to the saccharide portion. The chemical shifts of the two olefinic carbons (δ 124.7 and 144.1) and comparison of the remaining chemical shifts (Table 1) with those well established for pentacyclic triterpenes revealed the characteristic carbons of an olean-12-ene triterpene.5 The methylene resonance at δ 65.0 was diagnostic for the hydroxylated C-28, which correlated with two protons at δ 3.08 and 3.30 in the HMQC spectrum. The absence of a methylene resonance at high field (ca. δ 15.0)5,6 in the 13C NMR spectrum and the appearance of the signal at δ 4.14 in the 1H NMR spectrum, which correlated with C-28 in the HMBC spectrum, suggested a C-16-OH in 1. The correlations between H-16 and H-28a,b in the NOESY spectrum pointed to the R-configuration of C-16-OH. The lack of another methylene resonance at ca. δ 33.0 (C-22)6 and the
© 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 05/13/1999
874 Journal of Natural Products, 1999, Vol. 62, No. 6
Jiang et al.
Table 1. NMR Data of the Aglycon Moieties of Compounds 1-6 (400 MHz in CD3OD, J in Hz)a 1, 2b
a
position
δC
1
40.2
2
27.1
3 4 5 6
92.2 40.7 57.2 19.5
7 8 9 10 11 12 13 14 15
34.2 41.2 48.2 37.9 24.8 124.7 144.1 42.4 35.4
16 17 18 19
71.2 45.5 41.9 48.2
20 21
32.6 42.6
22 23 24 25 26 27 28
74.1 28.6 17.1 16.4 17.5 27.9 65.0
29 30 A (or B) 1 2 3 4 5 6 COMe COMe
33.7 25.4 169.9 130.1 138.2 16.1 21.1
3, 4b δH
δC
1.05 1.64 2.04 1.70 3.23
40.0 26.9
0.80 dd (11.0, 1.0) 1.60 1.42 1.35 1.70 1.90 5.37 m 1.75 1.33 4.14 m
92.0 40.5 57.0 19.2 33.8 41.1 48.2 37.8 24.6 125.9 143.1 42.2 31.7 74.1 44.7 41.5 48.2
2.50 1.10 2.53 1.55 2.29 5.47 dd (12.0, 5.6) 1.12 s 0.93 s 1.00 s 0.97 s 1.50 s 3.08 d (11.0) 3.30 d (11.0) 0.93 s 1.07 s
6.09 q (5.8) 2.00 d (5.8) 1.93 s
32.4 42.4 72.2 28.4 17.0 16.2 17.2 27.5 64.9 33.7 25.0
5, 6b δH
1.05 1.64 2.04 1.70 3.23 0.80 dd (11.0, 1.0) 1.60 1.42 1.35 1.70 2.02 5.42 m 1.86 1.50 5.24 m 2.60 1.24 2.36 1.68 1.80 5.45 dd (12.2, 5.8) 1.10 s 0.95 s 1.04 s 1.03 s 1.40 s 3.22 d (10.8) 3.36 d (10.8) 1.02 s 1.08 s
169.9 129.6 138.6 15.9 20.9
6.04 q (5.8) 2.00 d (5.8) 1.88 s
171.4 22.1
2.06 s
δC 40.0 26.9 92.0 40.6 57.0 19.3 34.0 41.0 48.1 37.7 24.6 124.6 143.9 42.2 35.2 71.8 45.3 41.7 48.1 32.5 42.4
δH 1.05 1.64 2.04 1.70 3.22 0.82 dd (11.0, 1.0) 1.60 1.42 1.36 1.68 1.90 5.36 m 1.73 1.35 4.14 m 2.49 1.08 2.52
33.6 25.2
1.55 2.29 5.45 dd (12.0, 5.6) 1.10 s 0.91 s 1.00 s 0.97 s 1.48 s 3.04 d (11.0) 3.30 d (11.0) 0.93 s 1.06 s
168.3 121.4 151.0 32.0 23.3 14.1
5.84 d (11.7) 6.28 dt (11.7, 7.2) 2.65 dt (7.2, 6.8) 1.46 m 0.97 t (7.2)
73.4 28.3 17.0 16.2 17.3 27.7 64.7
b
Assignments based on 2D NMR experiments. Data in the same column may vary slightly.
appearance of one proton at δ 5.47 (dd, J ) 12.0, 5.6 Hz), in combination with the resonances at δ 169.9, 138.2, 130.1, 21.1, and 16.1 in the 13C NMR spectrum, and δ 6.09 (1H, q, J ) 5.8 Hz), 2.00 (3H, d, J ) 5.8 Hz), and 1.93 (3H, s) in the 1H NMR spectrum, ascertained the presence of a angeloyl moiety rather than a tigloyl moiety in 1. Tigloyl methyl groups normally appear at higher field in the 13C NMR spectrum (ca. δ 13.0 and 15.0, respectively).4 The HMBC correlations between the proton at δ 5.47 and C-28, C-16 confirmed that the O-angeloyl group should be allocated to C-22. NOESY correlations between H-22 and Me-30,H-28a,b were observed, indicating the R-configuration of the O-angeloyl residue. The peak at δ 92.0 in the 13C NMR spectrum, showing a significant glycosidation shift, suggested linkage of the sugar moiety to the aglycon at C-3; H-3 (δ 3.23) correlated with Me-23 in the NOESY spectrum, indicating the usual β-configuration of the C-3-OH. The 13C NMR spectrum of the sugar portion of 1 exhibited signals for five anomeric carbons (δ 101.0, 101.0, 103.4, 103.9, and 105.9), one carbonyl carbon (δ 175.1), two methylene resonances (δ 62.9 and 63.1), and two methyl groups (δ 18.1 and 18.2). Five anomeric protons (δ 4.52,4.82,
5.00, 5.26, and 5.41) and two methyl doublets (δ 1.29 and 1.31, J ) 5.8) were observed in the 1H NMR spectrum of 1. These observations and comparison of the remaining chemical shifts with those reported in the literature7 revealed the presence of two rhamnoses, two galactoses, and one glucuronic acid in 1. This was confirmed by comparison with authentic sugars on co-TLC after acid hydrolysis. The 1H and 13C NMR chemical shift assignments of the sugars were made through 1H-1H COSY, HMQC, HMBC, HOHAHA, and NOESY experiments (Table 2). The positions of the glycosidic linkages were established from the connectivities indicated in the HMBC and NOESY spectra. In the HMBC spectrum, a cross peak between C-3 of the aglycon (δ 92.2) and H-1 of glucuronic acid (δ 4.52) indicated that glucuronic acid (glcA) was connected to C-3 of the aglycon. The linkage of the terminal galactose (galΙ) at C-2 of glcA was indicated by the cross peaks C-2glcA (δ 79.6)/H-1gal-Ι (δ 4.82), and C-1gal-Ι (δ 103.4)/H-2glcA (δ 3.93). Similarly, the linkages of the terminal rhamnose (rha-ΙΙ) at C-2 of the other rhamnose (rha-I), of rha-I at C-2 of the other galactose (gal-II), and of gal-II at the C-3 of glcA were indicated by the cross peaks H-1rha-II (δ 5.00)/ C-2rhaI (δ 79.6), C-1rha-II (δ 103.9)/H-2rha-I (δ 3.97),
Saponins from Maesa
Journal of Natural Products, 1999, Vol. 62, No. 6 875
Table 2. NMR Data of the Sugar Portions of Compounds 1-6 (400 MHz in CD3OD, J in Hz) 1, 3, 5b
2, 4, 6b
position
δC
δH
δC
δH
1 2 3 4 5 6 1 2 3 4 5 6
105.9 79.6 81.6 72.2 77.1 175.1 103.4 73.5 75.0 70.3 76.8 63.1
4.52 d (7.5) 3.93 4.11 3.62 3.68
105.9 80.0 81.3 72.2 72.0 175.0 102.8 76.2 78.4 72.7 78.1 63.7
4.50 d (7.5) 3.95 4.10 3.62 3.72
gal-II (gal)
1 2 3 4 5 6
101.0 76.8 76.0 72.2 76.6 62.9
rha-I
1 2 3 4 5 6 1 2 3 4 5 6
101.0 79.6 72.4 74.1 70.3 18.1 103.9 72.2 72.4 74.1 70.3 18.2
sugar glcA
gal-I (GLC)
rha-II
4.82 d (7.4) 3.59 3.50 3.85 3.70 3.62 3.90 5.26 d (6.7) 3.77 3.83 3.68 3.59 3.70 3.88 5.41 br s 3.97 3.83 3.40 4.14 1.29 d (5.8) 5.00 br s 4.01 3.72 3.40 3.72 1.31 d (5.8)
4.85 d (7.4) 3.27 3.38 3.12 3.40 3.55 3.88 5.22 d (7.0) 3.75 3.83 3.68 3.57 3.65 3.84 5.44 br s 4.00 3.82 3.42 4.12 1.30 d (5.8) 5.00 br s 4.02 3.72 3.40 3.72 1.31 d (5.8)
101.0 76.4 76.0 72.2 77.0 62.9 101.0 79.4 72.3 74.2 70.4 18.1 104.0 72.2 72.4 74.2 70.4 18.2
a Assignments based on 2D NMR experiments. same column may vary slightly.
b
Data in the
H-1rha-I (δ 5.41)/C-2gal-II (δ 76.8), C-1rha-I (δ 101.0)/ H-2gal-II (δ 3.77), H-1gal-II (δ 5.26)/C-3glcA (δ 81.6), and C-1gal-II (δ 101.0)/H-3glcA (δ 4.11). The NOESY experiment showed cross peaks that were used to define the interglycosidic linkages. Correlations between H-1 and H-5 of glcA, gal-I, and gal-II were observed, indicating β-configurations for these sugars. The JH-H values of the anomeric protons (7.5, 7.4, and 6.7 Hz, respectively) provided further evidence. On the basis of the foregoing, the structure of 1 was deduced as 3-O-{[(R-L-rhamnopyranosyl (1f2)-R-L-rhamnopyranosyl (1f2)-β-D-galactopyranosyl (1f3)]-[β-D-galactopyranosyl (1f2)]-β-D-glucuronopyranosyl}-22-O-angeloyl-camelliagenin A. Maelaxin B (2) showed, in the positive-ion FABMS and ESMS, a [M + 2Na - H]+ ion at m/z 1393 and a [M + Na]+ ion at m/z 1371. The negative-ion FABMS showed a fragmentation pattern similar to that of 1. Comparison of the 1H and 13C NMR data of both compounds indicated identical aglycon moieties. The main differences were the chemical shifts in the sugar portions. In the 1H NMR spectrum of 2, there were more signals in the region δ 3.05-3.45; in the 13C NMR spectrum, some of the carbon resonances (δ 78.3 and 78.1) were a little more downfield than those of 1 (δ 77.1 and 76.8). These observations indicated the replacement of one of the galactose units in 2 by a glucose, like a saponin previously isolated from Maesa ramentacea.2 This was confirmed by co-TLC with authentic samples after acid hydrolysis. Correlations in the HMBC and NOESY spectra indicated that the glucose was linked at C-2 of the glcA. The linkages of other sugars were the same as in 1. Thus, maelaxin B was defined as 3-O-{[(R-L-rhamnopyranosyl(1f2)-R-L-rham-
nopyranosyl(1f2)-β-D-galactopyranosyl (1f3)]-[β-D-glucopyranosyl (1f2)]-β-D-glucuronopyranosyl}-22-O-angeloylcamelliagenin A. Maelaxin C (3) and maelaxin D (4) showed, in the positive-ion ESMS, a [M + 2Na - H]+ ion at m/z 1435 and a [M + Na]+ ion at m/z 1413, 42 mass units higher than those of 1 and 2, and gave 13C and 13C DEPT NMR data consistent with a C67H106O30 molecular formula. Comparison of the 1H and 13C NMR data of the sugar portion of 3 and 4 with those of 1 and 2 indicated that 3 and 1, 4 and 2 had the same saccharide chains, respectively. Compounds 3 and 4 varied from 1 and 2 in the aglycon moieties. In the 1H NMR spectra of 3 and 4, an additional acetyl methyl singlet appeared at δ 2.06; a signal at δ 5.24 replaced the H-16 signal at δ 4.14. The 13C NMR spectrum of 3 and 4 showed upfield shifts of C-15 (-3.7 ppm) and C-17 (-1.1 ppm) and an additional resonance at δ 22.1, implying the acetylation of C-16-OH.6 Once again, the HMBC and NOESY experiments of 3 and 4 showed the same glycosidic linkages as in 1 and 2, respectively. The NOESY correlations H-16 (δ 5.24)/H-28a,b and H-22 (δ 5.45)/H-28a,b indicated the R-configurations of C-16-OAc and C-22-Oangeloyl. Thus, the structures of 3 and 4 were identified as 3-O-{[(R-L-rhamnopyranosyl (1f2)-R-L-rhamnopyranosyl (1f2)-β-D-galactopyranosyl (1f3)]-[β-D-galactopyranosyl (1f2)]-β-D-glucuronopyranosyl}-16-O-acetyl, 22-Oangeloyl-camelliagenin A and 3-O-{[(R-L-rhamnopyranosyl(1f2)-R-L-rhamnopyranosyl (1f2)-β-D-galactopyranosyl (1f3)]-[β-D-glucopyranosyl (1f2)]-β-D-glucuronopyranosyl}16-O-acetyl, 22-O-angeloyl-camelliagenin A, respectively. The positive-ion ESMS of maelaxin E (5) and maelaxin F (6) showed a [M + 2Na - H]+ ion at m/z 1407 and a [M + Na]+ ion at m/z 1385, 14 mass units higher than those of 1 and 2, corresponding to the molecular formula C66H106O29. The same saccharide chains as those of 1 and 2 were deduced for 5 and 6 by comparison of the NMR data. In the NMR spectra of 5 and 6, the signals of the angeloyl moiety were replaced by resonances at δC 168.3 (carbonyl), 151.0 (methine), 121.4 (methine), 32.0 (methylene), 23.3 (methylene), and 14.1 (methyl) and δΗ 6.28, 5.84, 2.65, 1.46, and 0.97. These observations indicated the existence of a 2-hexenoyl group instead of the angeloyl group in 5 and 6. This was confirmed by the 1H-1H COSY, HMQC, and HMBC experiments. The geometric configuration of the double bond was determined as Z from the JH-H value (11.7 Hz).8 A 2-(Z)-hexenoyl residue has also been found in the recently reported saponins from Maesa japonica.9 The NOESY correlations H-16 (δ 4.14)/H-28a,b and H-22 (δ 5.45)/H-28a,b indicated the R-configurations of C-16-OH and C-22-O-(2Z)-hexenoyl. Thus, the structures of 5 and 6 were established as 3-O-{[(R-L-rhamnopyranosyl (1f2)-RL-rhamnopyranosyl (1f2)-β-D-galactopyranosyl (1f3)]-[βD-galactopyranosyl (1f2)]-β-D-glucuronopyranosyl}-22-O(2Z)-hexenoyl-camelliagenin A and 3-O-{[(R-L-rhamnopyranosyl (1f2)-R-L-rhamnopyranosyl (1f2)-β-D-galactopyranosyl (1f3)]-[β-D-glucopyranosyl (1f2)]-β-D-glucuronopyranosyl}-22-O-(2Z)-hexenoyl-camelliagenin A, respectively. Finally, the absolute configurations of the sugars were determined as the D-form for glucuronic acid, glucose, and galactose, and the L-form for rhamnose by the method developed by Hara et al. (see Experimental Section).10 Maelaxins A-F (1-6) showed neither cytotoxic properties nor antitubulin activity. Experimental Section General Experimental Procedures. Optical rotations at 20 °C were taken on a Perkin-Elmer 241 polarimeter. Spectra
876 Journal of Natural Products, 1999, Vol. 62, No. 6
were recorded as follows: IR, Perkin-Elmer Spectrum BX FTIR system; NMR, Bruker AC 250 (13C NMR and DEPT spectra) and AMX 400 (1H and 2D NMR spectra). ESMS and FABMS were acquired on a VG-ZAB-SEQ spectrometer. Column chromatography: Si gel Merck H60. TLC: Si gel Merck 60 F254. Preparative HPLC: column, Waters Pre-Pak C18, 15 µm, 100 Å, 47 i.d. × 300 mm; eluent, MeOH-H2O-AcOH (70:30: 0.5); flow rate, 50 mL/min; UV detection (220 nm). Semipreparative HPLC: column, Waters Prep Nova-Pak C18, 6 µm, 60 Å, 25 i.d.× 100 mm; eluent, MeOH-H2O-AcOH (60:40: 0.1); flow rate, 6 mL/min; column, Beckman Ultrasphere ODS, 5 µm, 10 i.d.× 250 mm; eluent, CH3CN-H2O-AcOH (38:62: 0.5); flow rate, 3 mL/min; UV detection (220 nm). Analytical HPLC: column, Nova-Pak ODS C18, 3.9 i.d. × 150 mm; eluent, MeOH-H2O-AcOH (55:45:0.1), CH3CN-H2O-AcOH (33:67: 0.5); flow rate, 1 mL/min, UV and DR detections. GC: column, DB-5, J&W Scientific, 0.25 mm i.d. × 30 m; column temperature, 50-230 °C, 15 °C/min then 230 °C, 18 min; carrier gas, N2. Plant Material. Leaves of Maesa laxiflora Pitard were collected from Yen Chau (“son la”), Vietnam, in February 1996. Identification was confirmed by one of us (V. D.) and Tran Ngoc Ninh, Institut d’Ecologie, NCST, Hanoi, Vietnam. Voucher specimens (VN 065) are deposited at the herbarium of that institute. Extraction and Isolation. Dried leaves of M. laxiflora (1 kg) were extracted exhaustively with EtOH at room temperature. The EtOH extract was defatted with heptane and EtOAc and then partitioned with n-BuOH and H2O to give the n-BuOH extract (20.0 g). Repeated Si gel chromatographies eluted with CH2Cl2-MeOH-H2O from 30:10:1 to 12:6:1 gave a crude saponin fraction (4.0 g). Part of the crude saponin (1.8 g) was separated by preparative HPLC into fraction A (820 mg) and fraction B (165 mg). Semipreparative HPLC of 200 mg of fraction A (MeOH-H2O-AcOH, 60:40:0.1) gave 1 (73 mg, tR 73.2 min) and 2 (36 mg, tR 83.3 min). Semipreparative HPLC of fraction B (CH3CN-H2O-AcOH, 38:62:0.5) gave 3 (29 mg, tR 49.0 min), 4 (23 mg, tR 52.0 min), 5 (15 mg, tR 60.2 min), and 6 (24 mg, tR 65.4 min). Maelaxin A (1): white amorphous powder, mp 245-248 °C, [R]20D -24.6° (c 1.4, MeOH); IR (KBr) νmax 3426, 2925, 1681, 1636, 1384, 1081 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1393 [M + 2Na - H]+ (100), 1371 [M + Na]+ (82); positiveion FABMS m/z 1393 [M + 2Na - H]+ (100); negative-ion FABMS m/z 1347 [M - H]- (100), 1247 (6), 1201 (13), 1185 (9), 1055 (13), 893 (20), 793 (12), 731 (6), 555 (8). Maelaxin B (2): white amorphous powder, mp 242-245 °C, [R]25D -16.3° (c 1.5, MeOH); IR (KBr) νmax 3422, 2926, 1683, 1632, 1384, 1076 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1393 [M + 2Na - H]+ (44), 1371 [M + Na]+ (100); positive-ion FABMS m/z 1393 [M + 2Na - H]+ (100), 1371 [M + Na]+ (37), 1247 (13); negative-ion FABMS m/z 1347 [M - H](100), 1201 (13), 1185 (7), 1055 (20), 893 (45), 793 (5), 731 (10), 555 (6). Maelaxin C (3): white amorphous powder, mp 246-248 °C, [R]25D -18.2° (c 1.4, MeOH); IR (KBr) νmax 3427, 2924, 1716, 1634, 1384, 1073 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1435 [M + 2Na - H]+ (31), 1413 [M + Na]+ (100). Maelaxin D (4): white amorphous powder, mp 247-249 °C, [R]25D -15.6° (c 1.6, MeOH); IR (KBr) νmax 3425, 2926, 1710, 1635, 1384, 1074 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1435 [M + 2Na - H]+ (50), 1413 [M + Na]+ (100). Maelaxin E (5): white amorphous powder, mp 256-258 °C, [R]25D -5.6° (c 1.8, MeOH); IR (KBr) νmax 3420, 2926, 1698, 1622, 1384, 1074 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1407 [M + 2Na - H]+ (100), 1385 [M + Na]+ (20). Maelaxin F (6): white amorphous powder, mp 258-260 °C, [R]25D -10.0° (c 1.4, MeOH); IR (KBr) νmax 3419, 2927, 1700, 1620, 1385, 1075 cm-1; 1H and 13C NMR, Tables 1 and 2; ESMS m/z 1407 [M + 2Na - H]+ (77), 1385 [M + Na]+ (100).
Jiang et al.
Acid Hydrolysis of 1. Compound 1 (2 mg) was refluxed with 7% HCl (1 mL) for 3 h. The reaction mixture was partitioned between EtOAc and H2O. The H2O layer was concentrated and checked by TLC (Si gel), together with the authentic sugar samples (CH2Cl2-MeOH-H2O, 6.5:4.0:0.8; Rf, 0.20 for glcA, 0.60 for gal, 0.76 for rha). Acid Hydrolysis of 2. Compound 2 (2 mg) was refluxed with 7% HCl (1 mL) for 3 h. The reaction mixture was partitioned between EtOAc and H2O. The H2O layer was concentrated and checked by TLC (Si gel) together with the authentic sugar samples (CH2Cl2-MeOH-H2O, 6.5:4.0:0.8); Rf, 0.2 for glcA, 0.64 for glc, 0.60 for gal, 0.76 for rha). Determination of the Absolute Configuration of Sugars. A mixture of 1 and 2 (5 mg) was hydrolyzed to give the sugar mixture, which was methylated with trimethyldiazomethane in MeOH. After being dried, the residue was dissolved in MeOH (2 mL) and treated with NaBH4 (20 mg) at room temperature for 30 min. The mixture was passed through a Waters SEP-PAK C18 cartridge column (Milford, MA) eluted with H2O (5 mL) and then MeOH-H2O (1:1, 5 mL). The MeOH-H2O (1:1) eluent was dried in vacuo and then dissolved in dry pyridine (1 mL), and 3 mg l-cysteine was added. The mixture was heated at 60 °C for 2 h, dried in vacuo, and trimethysilylated with N,O-bis(trimethylsilyl)acetamide (0.1 mL) at 70 °C for 0.5 h. After partition between heptane and H2O, the heptane extract was analyzed by GC. The sugar derivatives showed tR of 19.62, 24.04, and 24.93 min, identical to those of authentic L-rha, D-glc, and D-gal, respectively. Under the same conditions, derivatives of L-glc and L-gal showed tR of 25.01 and 26.18 min, respectively. Bioassays. The cytotoxicity test against A-549 cell line (NCI) was performed using a standard MTT-based colorimetric assay.11 Tubulin assembly assay: the tubulin solution was prepared and the polymerization was initiated (by a temperature shift from 6 to 37 °C) as described previously.12 The polymerization rate was calculated from the slope of the increase of turbidity measured as a function of time.12 The indicated concentration (10 mg/mL) of the inhibitor added to the cold (6 °C) tubulin solution decreased the rate by 41%. The control used for comparison is colchicine (rate decrease: 50% at 1 µg/mL). Acknowledgment. Thanks are expressed to V. Guerineau for his assistance in HPLC and to B. Hill, Centre de Recherche Pierre Fabre, Castres, France, for the biological assay results. One of the authors (Z.J.) expresses his gratitute to Dr. P. Potier, ICSN, CNRS, Gif-sur-Yvette, France, for fellowship support. References and Notes (1) Sindambiwe, J. B.; Calomme, M.; Geerts, S.; Pieters, L.; Vlietinck, A. J.; Vanden Berghe, D. A. J. Nat. Prod. 1998, 61, 585-590. (2) Tuntiwachwuttikul, P.; Pancharoen, O.; Mahabusarakam, W.; Wiriyachitra, P.; Taylor, W. C.; Bubb, W. A.; Towers, G. H. N. Phytochemistry 1997, 44, 491-495. (3) Chakravarty, A. K.; Das, B.; Pakrashi, S. C. Phytochemistry 1987, 26, 2345-2349. (4) Yoshikawa, K.; Taninaka, H.; Kan, Y.; Arihara, S. Chem. Pharm. Bull. 1994, 42, 2023-2027. (5) Tori, K.; Seo, S.; Shimaoka, A.; Tomita, Y. Tetrahedron Lett. 1974, 4227-4231. (6) Mahato, S. B.; Kundu, A. P. Phytochemistry 1994, 37, 1517-1575. (7) Sindambiwe, J. B.; Balde´, A. M.; De Bruyne, T.; Pieters, L.; Van Den Heuvel, H.; Claeys, M.; Vanden Berghe, D. A.; Vlietinck, A. J. Phytochemistry 1996, 41, 269-277. (8) Jackman, L. M.; Sternhell, S. In Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry, 2nd ed.; Pergamon: Oxford, UK, 1969; Part 4, p 302. (9) Koike, K.; Kudo, M.; Jia, Z.; Nikaido, T.; Ide, Y.; Sakura, T. J. Nat. Prod. 1999, 62, 228-232. (10) Hara S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501507. (11) Mosman, T. J. Immunol. Methods 1983, 65, 55-63. (12) Zavala, F.; Gue´nard, D.; Potier, P. Experientia 1978, 34, 1497-1499.
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